Focused on elucidating the mechanisms of evolution at the molecular and population-genetic levels, this project involves an integration of theory development and experimental work performed in a phylogenetic comparative manner. The molecular/cellular level focus is on the rate of error production via DNA replication, mRNA transcription, and protein translation in ~50 bacterial and eukaryotic species. This work seeks to test the drift-barrier hypothesis, which postulates that the level of refinement that natural selection can achieve with any trait is limited by the power of random drift and inversely proportional to the effective population size. Measures of replication-fidelity derive from mutation accumulation in long-term sets of serially bottlenecked replicate lines followed by whole-genome sequencing. Newly developed methods will yield parallel estimates of the much higher rates at which inappropriate ribonucleotides appear in mRNAs and erroneous amino acids are incorporated into proteins, allowing evaluation of whether negative associations exist between error propagation at these two levels. The population-genetic mechanisms of evolution will be clarified via a project to sequence 5000 genomes from geographically widespread isolates of the model microcrustacean Daphnia pulex, as well as from smaller numbers of key outgroup species. Combined with information on the fine-scaled pattern of recombination, this study will reveal the relative magnitudes of drift, mutation, and recombination in a collection of ~60 populations, a survey far beyond that for any other species. This will enable a test of the hypothesis that variation at the level of gene structure and genomic architecture is directly driven by the local population-genetic environment. The D. pulex system also has unique features for gaining insights into two major unsolved mysteries in evolutionary genetics: the origin and long-term effects of introns in protein-coding genes, and the causes and consequences of the loss of meiotic recombination. Relevance to human health. Because mutation is the source of all variation upon which natural selection acts, provides the fuel for the emergence of pathogens, and is the ultimate cause of genetic disorders and cancer, our work on replication fidelity has broad significance for diverse human-health issues. Because transcription and translation errors lead to cellular toxicity and protein aggregation, work at these levels also has significant applied implications. Finally, for the first time, the 5000 Genomes Project will reveal how population evolutionary features are defined by the relative power of drift, mutation and recombination, yielding insight into the factors driving the efficiencies and mechanisms by which all species respond to natural selection. Elucidation of the molecular/cellular mechanisms by which meiotic production of haploid eggs requiring fertilization is converted to ameiotic production of diploid eggs is highly desirable as it would open up possibilities of clonal propagation in diverse species.